Induced pluripotent stem cells (iPSCs) can be generated by retroviral overexpression of the transcription factors Oct4, Sox2, Klf4, and c-Myc, but integration of foreign DNA can cause genomic dysregulation. Introducing these transcription factors as cell-permeant proteins (CPPs) avoids such issues, but the efficiency of iPSC generation by this method is low. In an effort to improve the efficiency of CPP-based reprogramming, Lee et al. uncovered a link between innate immunity and the induction of pluripotency. They compared the gene expression profiles of human fibroblasts subjected to either retroviral- or CPP-mediated delivery of Sox2 or Oct4. Within the first 48 hours, abundance of mRNAs of Sox2 or Oct4 target genes and pluripotency-associated genes increased in response to retroviral-mediated delivery but not CPP-mediated delivery, indicating a delay in target and pluripotency gene expression with the CPP method. CPP-mediated delivery of Sox2 or Oct4 concomitant with retroviral delivery of a control plasmid (or a nonintegrating mutant) recapitulated the gene expression profile of retroviral delivery of Sox2 or Oct4. Because viral infection activates innate immunity through Toll-like receptors (TLRs), the authors investigated the effects of inhibiting components of TLR-signaling pathways with inhibitory peptides or short hairpin RNA. Inhibition of TLR3 or its adaptor TRIF, but not components of other TLR pathways, ablated the increase in Oct4 target gene and pluripotency gene expression elicited by retroviral delivery of Oct4 and resulted in fewer and smaller colonies of pluripotent cells than controls. The TLR3 agonist poly-inosinic-polycytidylic acid (Poly I:C) reproduced the enhancement of Oct4 target gene and pluripotency gene expression and the number and size of the resulting pluripotent cell colonies caused by retroviral delivery. Chromatin immunoprecipitation revealed hallmarks of epigenetic modification indicating an open chromatin state at the promoters of Oct4 or Sox2 after CPP delivery with TLR3 activation induced by Poly I:C or control retroviral vector in human fibroblasts and mouse embryonic fibroblasts (MEFs). Knockdown of TLR3 effectors IKKε, NF-κB, or IRF3 inhibited the enhancement of Oct4 target gene expression induced by coapplication of Poly I:C with CPP-mediated delivery of Oct4, as well as the increase in modifications that promote open chromatin at the Oct4 promoter in MEFs. Downstream effects of viral infection through the proinflammatory TLR3 pathway thus pave the way for reprogramming cells with delivered transcription factors (see commentary by O’Neill).